Peptide or protein-capturing surfaces for high throughput MALDI mass spectrometry

ABSTRACT

Methods and compositions useful for capturing proteins or peptides from a sample mixture and analyzing the captured proteins or peptides via high throughput electrospray ionization (ESI) or matrix-assisted laser desorption/ionization mass spectrometry (HT MALDI MS) are provided. The methods and compositions are useful in large scale, simultaneous analyses of proteins and peptides present in a cell(s), tissue(s), or biological fluid(s).

FIELD OF THE INVENTION

The present invention relates to methods and compositions useful forcapturing proteins or peptides from a sample mixture and analyzing thecaptured proteins or peptides via high throughput electrosprayionization (ESI) or matrix-assisted laser desorption/ionization massspectrometry (HT MALDI MS). The subject methods and compositions areuseful in large scale, simultaneous analyses of proteins and peptidespresent in a cell(s), tissue(s), or biological fluid(s).

BACKGROUND OF THE INVENTION

The inception of the now completed Human Genome Project has spurred anexplosion in genomic research. DNA expression arrays, and other genomictools were created to take advantage of the wealth of informationpromised, and currently provided by a completely sequenced human genome.As a result, genomics has come to dominate the biological landscape.Genomics, however, has its limits. Now more than ever, it is becomingclear that gene sequence alone cannot predict the fate or behavior ofprotein products. Though high throughput transcriptional profiling onDNA chips or microarrays can be used to determine when, where, and howmuch RNA is transcribed, there is rarely a relationship between RNAtranscription and protein expression.^(1,2,3) Post transcriptionalmodifications such as phosphorylation, and mRNA and peptide degradationcan alter the function and concentration of a protein and are invisibleto transcriptional profiling.

This gap between genomics and cellular behavior impedes elucidation ofprotein function as well as the identification of novel drug targets.Research in the post genomic era, then, will require a bridge betweengenomics and cellular behavior. Proteomics, which goal is to study theprotein products of the genome, promises to be this bridge. By takingadvantage of newly developed analytical tools, proteomics strives tostudy the dynamic, protein equivalent of the genome, the proteome. In sodoing, proteomics would provide fundamental information on the molecularworkings of the cell, as well the ability to observe the effect thatspecific diseases or drug treatments have on protein cascades.⁴ Indeed,the potential ability of proteomics to provide information aboutproteins on a global scale is tremendous for pharmaceutical research, asover 75% of the predicted proteins in multicellular organisms have noknown cellular function⁵ and as many as 4,000 protein drug targets arepredicted.⁶

Fundamental to proteomics are the abilities to both separate andidentify proteins. Currently separation is achieved via several methods,the most popular being two dimensional gel electrophoresis (2-DE).⁷Separation by 2-DE is on the basis of charge (isoelectric focusing) andsize (PAGE). Gels are stained by a variety of methods, includingCoomassie blue stain, silver stain, fluorescent dyes, and radio labels.Once the gel is stained, proteins are easily located and excised fromthe gel. Currently, the most effective method for protein identificationis by mass spectrometry (MS). The sensitivity of mass spectrometry,coupled with recently developed ionization methods, allows for accurateprotein characterization and identification. Indeed, advancements inmass spectrometry are responsible for the growth of proteomics as afield.

The now inseparable relationship between mass spectrometry andproteomics can be traced to two technological breakthroughs in the late1980's: electrospray ionization (ESI) and matrix-assisted laserdesorption/ionization (MALDI).^(8,9,10) In contrast to previousionization methods, both ESI and MALDI are “soft” ionization methods,capable of generating ions from proteins and peptides withoutsignificant fragmentation. So soft are ESI and MALDI as ionizationmethods, that under certain conditions even non-covalent interactionsare undisturbed, allowing for the analysis of large multi-proteincomplexes.¹¹ ESI is easily coupled to liquid phase chromatographic andelectrophoric techniques, a quality for which the method quickly gainedpopularity. Due to this, and the method's tendency to produce multiplecharged ions allowing detection outside the nominal m/z range of simpleinstruments, ESI soon became the method of choice for analysis ofproteins in the liquid phase.^(12,13)

Ions produced via MALDI are, in contrast, largely singly charged,providing mass spectra that are easily interpreted. Furthermore, thetime-of flight (TOF) mass analyzer to which MALDI is most often coupledis robust, simple, sensitive, and capable of detecting proteins as largeas 100,000 mass units (amu).^(14,15) Both methods are now established asstate of the art analytical tools in proteomics, finding applications inprotein identification by mass mapping, and single peptidefragmentation, as well as the identification and characterization ofpost-translation modifications such as protein phosphorylation.¹³Perhaps the most popular of these applications is protein identificationby mass mapping, in which proteins, once separated by 2-DE or HPLC, aredigested by a sequence-specific proteolytic enzyme such as trypsin. Upondigestion by such an enzyme, a specific protein will produce a uniqueset of polypeptide sequences, which upon detection and analysis by MS,yields a polypeptide mass-map. This mass-map, which is unique, can beused to identify the protein. Peptide mass-mapping has been used in theproteomic analysis of Haemophilus influenzae as well as several strainsof yeast used in the brewing industry.¹⁶ Mass spectrometry is also usedfor protein sequencing, replacing Edman sequencing. Mass spectrometryallows for the analysis of subfemtomole quantities and is not restrictedby N-terminal modifications, both problems associated with theEdman-based method.¹⁷

Neither ESI nor MALDI, however, are without limitations. Due to theattomolar sensitivity of both ESI and MALDI, and the small volume of MSsamples, cleanliness of biological samples is perhaps the greatestlimitation for proteomic MS.^(18,19) This limitation is particularlynoticeable in proteomics as protein samples obtained from 2-DE geldigestion contain salts, ionic detergents and involatile solvents thatcan greatly reduce signal intensity and resolving power.²⁰ Theimportance of sample cleaning and preparation is in fact so great thatthe literature abounds with novel sample prep protocols, particularlyfor the more popular MALDI method.^(21,22,23)

Furthermore, the emergence of products specifically targeted at samplepreparation for proteomic MS highlights the significance of this step.Millipore (Bedford, Mass.) has introduced the ZipTip™, a 10 μl pipettetip packed with a polymer stabilized expanded bed (PSEB) of C18 reversephase chromatographic media. When a protein or peptide sample isintroduced into the pipette, peptides are captured by the C18 media viahydrophobic interactions. The sample can then be washed of impuritiesand subsequently eluted.¹⁹ Sample cleaning with ZipTip™ technology hasbeen shown to significantly improve the signal-to noise ratio of peptidemass maps obtained from MALDI-TOF.²²

Sample cleaning has also been achieved by assembling reverse-phase mediadirectly onto the MALDI probe. A stainless steel MALDI probe is coatedwith gold, and then self-assembled monolayers (SAMs) are created bytaking advantage of gold-thiol chemistry. At present, both C18 and ionicreverse phase media have been created.^(23,24) Although both of thesemethods achieve sample cleaning and improve spectrum quality, theynonetheless fail to do so in high throughput fashion. Proteomics is ahigh throughput field and the need to individually purify and analyzesamples with MS is severely limiting.⁷ With this in mind, Bruker(Bremen, Germany) has designed the AnchorChip, a MALDI target with 384individual hydrophilic anchors. The AnchorChip is stainless steel with ahydrophobic coating that allows for protein and peptide capture. Samplesare spotted with a water insoluble matrix such as α-cyano4-hydroxycinnamic acid (HCCA), and then washed. Samples are thenre-dissolved and allowed to crystallize, whereupon they shrink to thehydrophilic anchor.²⁵ Despite allowing for high throughput in samplepreparation and MS analysis, the anchor chip is cost prohibitive, andrequires stringent washing prior to re-use. Furthermore, the presence of384 liquid targets on a flat surface increases the possibility ofcross-contamination.

In order to eliminate the bottle-neck of proteomic sample preparation, ahigh throughput tool is required. Ideally, this tool should be bothinexpensive and disposable. Most importantly, the tool should reducesample cross-contamination. The present invention overcomes problems inthe art by providing such a tool.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods useful inperforming high throughput analysis of multiple protein and/or peptidesamples. The compositions comprise a solid support having a plurality ofcompartments, wherein one or more of the compartments has a specificaffinity layer attached thereto for capture of the peptides or proteinspresent in the samples. Preferably, more than one of the compartmentshas a specific affinity layer attached thereto. Even more preferably,all or almost all of the compartments have a specific affinity layerattached thereto.

The solid support may be comprised of any material as long as a specificaffinity layer for capturing peptides or proteins may be attachedthereto. Preferably, the solid support is comprised of a polymer such aspolypropylene, cyclic olefin copolymer, polyethylene, polymethylmethacrylate, polyvinyl chloride, polymethyl pentene, polycarbonate,polysulfone, polystyrene copolymer, poly(styrene-co-maleic anhydride,polypropylene copolymer, fluoropolymer, or polyamide.

Conveniently, many readily available materials may be used inconfiguring the solid supports of the present invention. Examplesinclude dishes, slides, strips or plates. Preferably, microwell platesor microtiter dishes are used.

Many different materials may be used as the specific affinity layer.Examples include hydrophobic surface layers, ionic surface layers, ormetal chelating surface layers. Preferably, a hydrophobic surface layeris used. Even more preferably, the hydrophobic surface layer maycomprise a long chain aliphatic, such as octadecyl vinyl ether (ODVE),octadecyl amine. (ODAM), tri(ethylene glycol)-methyl-vinyl-ether (TMVE),stearyl alcohol (SA), nonylphenoxypoly(ethyleneoxy)ethyl-methacrylate(C9 poly), or stearyloxypolyethyleneoxy-ethyl-methacrylate (SPMA).Specific species, such as octadecyl vinyl ether (ODVE) or octadecylamine (ODAM), may be more preferred.

Various combinations of specific affinity layers and other surfaces maybe used in the subject solid supports. For example, a specific affinitylayer may be attached to a first portion of the one or more compartmentsand a non-binding surface may be located in a second portion of the oneor more compartments. Alternatively, the one or more compartments havinga specific affinity layer attached thereto may further have ahydrophilic area devoid of and surrounded by the specific affinitylayer.

In still another embodiment, the solid support may have a specificaffinity layer attached to a first portion of the one or morecompartments, wherein the first portion of the one or more compartmentscomprises a hydrophilic area devoid of and surrounded by the specificaffinity layer attached thereto and may further have the one or morecompartments comprise a non-binding surface located in a second portionthereof.

The present invention also provides a method of performing highthroughput analysis of multiple samples, each of said samples comprisinga mixture of peptides or proteins. The methods employ use of a subjectsolid support. An example of such a method comprises the steps of:

(a) providing a solid support comprising a plurality of separatecompartments, wherein one or more of the compartments has a specificaffinity layer attached thereto for capture of the peptides or proteinspresent in the samples;

(b) applying each of said samples or an aliquot thereof, to a separatecompartment having the specific affinity layer attached thereto;

(c) washing the peptides or proteins which have been captured by thespecific affinity layer, and

(d) performing high throughput electrospray ionization (ESI) ormatrix-assisted laser desorption/ionization (MALDI) mass spectrometryanalysis on the captured peptides or proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts in a schematic the major steps of the present invention.A sample comprising a mixture of peptides or proteins is added to aseparate compartment on or in a solid support having a specific affinitylayer attached thereto. The specific affinity layer captures thepeptides or proteins in the sample. The captured proteins are thenwashed and may be analyzed via MALDI MS as represented by the MSspectrum.

FIG. 2A shows the results of protein capture on non-treated polystyrene.

FIG. 2B graphically depicts the degree of protein attachment at varyingconcentrations of ODVE on coated plates as a function of plasmatreatment time.

FIGS. 3A-3C show an ODVE coated plate to which a BSA digest was appliedand processed (Figs. A-C showing different sample sizes).

FIGS. 4A-4F show a comparison of MALDI TOF spectra for a gold depositedplate (A-C showing different sample sizes) and an ODAM coated plate,both of which had a BSA digest applied and processed (A-C showingdifferent sample sizes).

FIG. 5 schematically illustrates a compartment of a solid support havingan affinity layer (C18) deposited near the bottom of the compartment anda non-binding surface located near the upper portions of thecompartment.

FIGS. 6A and 6B schematically illustrate a microwell plate and a singlewell of the same microwell plate. The dark oval in the single wellrepresents a hydrophilic spot surrounded by an affinity layer, e.g., C18(6B). Multiple wells, each having a hydrophilic spot surrounded by anaffinity layer are illustrated in the microwell plate (6A).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for use in largescale, simultaneous processing of proteins and/or peptides present in asample such as a cell(s), tissue(s) or biological fluid(s). The subjectcompositions and methods overcome current limitations in the art byallowing most of the major steps involved in proteomic processing of asample to be performed at a single location on a solid support orsubstrate.

In accordance with the present invention, there are providedcompositions comprising a solid support or substrate having a pluralityof compartments for sample application and processing. At least onecompartment has a specific affinity layer attached thereto for proteinand/or peptide capture. As used herein “compartment” or “compartments”refers to any separate area or space, which is physically separated byother areas or spaces. Thus, examples of compartments include but arenot limited to wells, indentations, chambers, sections, or slots.

Preferably, more than one compartment has a specific affinity layerattached thereto for protein and/or peptide capture. Even morepreferably, all or almost all of the compartments have a specificaffinity layer attached thereto for protein and/or peptide capture.

The terms “solid substrate” and “solid support” are used interchangeablyherein and refer to any apparatus having multiple compartments intowhich a specific affinity layer may be attached. Thus, a solid supportor substrate for use in the present invention may take any number offorms, so long as a specific affinity layer may be attached thereto. Asused herein, “plurality” refers to more than one. The term “peptides orproteins” is meant to include all proteinaceous materials includingpeptides, polypeptides, and full-length proteins as well as fragmentsand combinations thereof. Hence, the term “peptides or proteins” is usedherein in its broadest sense. The term, however, can also refer to asample comprising proteinaceous material in the form of peptides only,or proteinaceous material in the form of full-length proteins only, orproteinaceous material in the form of fragments of full-length proteins,or any and all combinations thereof.

As described above, in order to reduce the possibility of crosscontamination of samples on and/or within the solid support, thereshould be provided therein or thereon compartments such as separatewells, indentations, chambers, or slots for sample application.Different features may be built into the compartments such as e.g.,porous surfaces or rough surfaces to increase surface area and thebinding capacity thereof.

The solid support may be any material that is capable of binding orholding the specific affinity layer. For example, glass, plastic,silicones, and elastomers, including silicone, hydrocarbon, andfluorocarbon elastomers may all be used. Ceramic, gold, stainless steel,aluminum, or silicon wafers may also be used as a support. If a metalsupport is used, the surface thereof may be coated with a material thatallows derivatization of the surface. A metal surface may be coated withe.g., silicon oxide, titanium oxide or gold. Preferably, a polymer isused to compose the solid support. Examples of polymers include but arenot limited to polypropylene, polystyrene, polyethylene, polymethylmethacrylate, polyvinyl chloride, polymethyl pentene, polycarbonate,polysulfone, polystyrene copolymers such as poly(styrene-co-maleicanhydride) (SMA) and ABS, polypropylene copolymers, cyclic olefincopolymer, fluoropolymers, and polyamides. In a more preferredembodiment, polystyrene is used.

In accordance with the present invention, it has been surprisinglydiscovered that high quality mass spectrometry (MS) spectra may beobtained for proteins captured on modified polymer surfaces. Thus,polymers, which can be easily and inexpensively manufactured in a widevariety of shapes, are preferred materials for the solid support. Plasmatreatment may be used to alter the surface chemistry of polymers.Methods of plasma treatment with inert gases to alter the surfacechemistry of polymers are well known and are described in the literatureincluding Sharpe, L. H, and Schonhorn, J., Adv. Chem. Ser. (1964)4:189-201; Hansen, R. H., and Schonhom, J. (1966) J. Polym. Sci. B,Polym. Lett. Ed. 4:203-209; and Sheu, M. S. et al., (1993) J. AdhesionSci. Tech. 7:1065-1076, the disclosures of which are incorporated byreference herein as if full set forth. Plasma coating of polymermicroplates as a method is relatively facile, and therefore allows forinexpensive, high-volume manufacture. Preferably, argon plasma treatmentis used as described in Examples 1 and 2.

Readily available materials may be used in configuring the solidsupports in accordance with the present invention. Examples of materialsto which an affinity layer may be applied and which may serve as solidsupports include but are not limited to dishes, slides, strips, orplates such as for example, polymer injection molded plates, polymerglass bottom plates, gold deposited plastic plates, microscope slides,microwell plates, microtiter dishes, and microplates. Examples ofpreferred materials include microwell plates and microtiter dishes.

Examples of specific affinity layers which may be used in the methodinclude but are not limited to hydrophobic surface layers, ionic surfacelayers, or metal chelating surface layers.

Examples of hydrophobic surface layers for use in the present inventioninclude long chain aliphatic hydrocarbon layers such as e.g., C1 to C18aliphatic hydrocarbons. Examples of long chain aliphatic hydrocarbonswhich may be used as specific affinity layers include but are notlimited to octadecyl vinyl ether (ODVE), octadecyl amine (ODAM),tri(ethylene glycol)-methyl-vinyl-ether (TMVE), stearyl alcohol (SA),nonylphenoxypoly(ethyleneoxy)ethyl-methacrylate (C9 poly), orstearyloxypolyethyleneoxy-ethyl-methacrylate (SPMA).

Examples of ionic surface layers, which may be attached to a solidsupport, include anionic or cationic surface layers. Examples of anionicsurface layers include sulfate anion layers and carboxylate anionlayers. Examples of cationic surface layers for use in the presentinvention include secondary, tertiary, or quaternary amine layers.

An example of a metal chelating surface layer is nickel. Otherappropriate metal chelating surfaces include copper, cobalt, zinc, iron,aluminum and calcium.

Multiple samples, each sample comprising a mixture of peptides orproteins, may be applied to a solid support and each sample may beapplied to a separate compartment, on or within the solid support. Thus,peptides or proteins present in an individual sample may be captured andadsorbed to an individual compartment having a specific affinity layerattached thereto. This allows simultaneous processing of multiplesamples comprising peptides or proteins captured by the specificaffinity layer on the solid support. Each sample applied to acompartment may be identified by a numbered ordered arrangement astypically found on microtiter dishes and microwell plates. For example,the solid support may contain an array of horizontal and/or verticalrows of compartments, which form a regular geometric pattern such as asquare, rectangle, or circle.

Microwell plates, e.g., those having 96 or 384 wells, are widely useddisposable tools for high throughput (HT) drug screening and samplepreparation and may be conveniently used in the compositions and methodsof the invention. The specific affinity layer may be directly orindirectly deposited preferably to the bottom surface of each individualwell, prior to applying the sample comprising peptides or proteins.

As demonstrated schematically in FIG. 1, a specific affinity layer maybe attached to the bottom and/or inside walls of the wells of amicrowell plate which can be used to capture peptides or proteins from acomplex mixture. After washing and desalting, the surface capturedpeptides or proteins can be extracted for MALDI MS analysis.

In one embodiment, the solid support may be a microwell plate. In orderto prepare the surface of a hydrophobic C-18 affinity plate, aninjection molded microwell plate may be prepared withanhydride-containing copolymers such as poly(styrene-co-maleicanhydride) (SMA). 1-Octadecylamine (ODAM) reacts directly with anhydridecopolymers to form a C18 surface. Lee, K. I., (1996)Styrene-Maleic-Anhydride Copolymer. In Polymeric Materials Encycl.Salamone (ed.), Vol 10, pp. 8010-14.

In another embodiment, glass slides or glass bottom plates may beemployed. In order to prepare a glass bottom plate or slide, well-knownmethods of silane chemistry may be applied to modify the glass bottoms.Polypropylene plates filled with 50% glass spheres may also be modifiedusing such methods. These supports may be modified with anoctadecenetrimethoxysilane alcohol solution to form bonded C18 on theplate bottoms or slide surfaces.

In still another embodiment, a gold deposited plate may be used. Inorder to prepare a gold deposited plate, a C-18 self-assembled monolayer(SAM) surface can be prepared on the gold deposited plate bottom byapplying 1-octadecenethiol alcohol solution (ODAM) as described in theexamples.

In yet another embodiment, octadecyl vinyl ether (ODVE) may be used as aspecific affinity layer on the solid support. ODVE may be dissolvedpreferably in isopropyl alcohol in a concentration in a range of fromabout 0.1 to 1.0 weight percent. The ODVE solution may be applied to thesolid support, allowed to dry and then exposed to plasma treatment.

One skilled in the art may select different specific affinity layers foruse in configuring a solid support based on the ability of the specificaffinity layer to capture peptides and proteins. The literature isreplete with assays, which may be used to measure levels of proteinbinding. Such assays include but are not limited to a colloidal goldassay and a fluorescein isothiocyanate-tagged BSA (FITC-BSA) assay.Preferably, an FITC-BSA assay is used.

In accordance with the present invention, it may be desirable to purifydilute samples and concentrate the captured peptides or proteins onto aspecific area of a compartment on or within the solid support. Thus inanother embodiment, there is provided a solid support for use inperforming high throughput analysis of multiple samples, each of saidsamples comprising a mixture of peptides or proteins, said solid supportcomprising a plurality of compartments for sample application. One ormore compartments has a specific affinity layer attached to a firstportion of the compartment and further has a non-binding surface locatedin a second portion of the same compartment. By “non binding surface” ismeant a surface which is low binding for proteins. Such surfaces may beproduced following the methods described in U.S. Pat. Nos. 6,093,559 and6,319,664, the disclosures of which are incorporated by reference hereinas if fully set forth. For example, a specific affinity layer may beapplied to the bottom surface of one or more compartments, while anon-binding surface may be created on other portions of the samecompartment(s) (e.g., the sides or walls of the compartment(s), or theupper sides or walls of the compartment(s)) (FIG. 5). When a diluteprotein digest solution is loaded into such a compartment(s), peptidesand/or proteins will not only be captured but will also be condensed onthe surface having the specific affinity layer attached thereto. Afterdesalting, the captured peptides can be recovered with very small volumeof eluant (such as an acetonitrile solution) and loaded onto MALDI MStarget plates.

In another embodiment, the solid support may be configured so that thedesalting step and matrix spotting process can be combined, thusreducing the number of transfer steps. The present invention thereforefurther provides a solid support for use in performing high throughputanalysis of multiple samples, each of said samples comprising a mixtureof peptides or proteins, said solid support comprising a plurality ofcompartments for sample application. One or more compartments has aspecific affinity layer attached thereto for capture of the peptides orproteins present in the samples and the one or more compartments havinga specific affinity layer attached thereto further has a hydrophilicarea devoid of and surrounded by the specific affinity layer. Forexample, after attachment of a specific affinity layer to one or morecompartments, a small hydrophilic spot can be created amidst thespecific affinity layer by removing a portion of the specific affinitylayer using a physical method such as laser ablation (FIG. 6). When aprotein digest solution is loaded into such a compartment, peptides willbe captured onto the specific affinity layer. After desalting, thecaptured peptides can be recovered by an eluant or wash solution (e.g.,acetonitrile) and then may be co-crystallized with matrix on thehydrophilic area without further transfer to another target plate. Thisaspect of the invention further reduces loss of sample due to transfersteps.

Of course, the solid support may combine various features describedabove. For example, one or more compartments having a specific affinitylayer attached to a first portion of the compartment(s) may further havea hydrophilic area devoid of and surrounded by the specific affinitylayer. The same compartment or compartments may also have a non-bindingsurface located in a second portion thereof.

In another aspect of the invention, there is provided a method forperforming high throughput analysis of multiple samples, each of saidsamples comprising a mixture of peptides or proteins. In accordance withthe method, individual samples may be simultaneously processed atdifferent locations (compartments) on the solid support. The methodcomprises the steps of: (1) providing a solid support with a pluralityof separate compartments for sample application, wherein one or more ofthe compartments has a specific affinity layer attached thereto forcapture of the peptides or proteins present in the sample; (2) applyinga sample or an aliquot thereof to a separate compartment having aspecific affinity layer attached thereto; (3) washing the peptides orproteins which have been captured by the specific affinity layer, and(4) performing high throughput electrospray ionization (ESI) ormatrix-assisted laser desorption/ionization mass spectrometry (HT MALDIMS) analysis on the captured peptides or proteins. Preferably, highthroughput matrix-assisted laser desorption/ionization time of flightmass spectrometry (HT MALDI TOF MS) analysis is performed.

In a further embodiment, the method comprises the steps of: (1)providing a solid support with a plurality of compartments for sampleapplication, one or more of said compartments having a specific affinitylayer attached thereto for capture of the peptides or proteins in thesample, wherein the one or more compartments having a specific affinitylayer attached thereto has the layer attached to a first portion of theone or more compartments and further has a non-binding surface locatedon a second portion of the same compartment(s); (2) applying each ofsaid samples or an aliquot thereof to a separate compartment having thespecific affinity layer attached thereto; (3) washing the peptides orproteins which have been captured by the specific affinity layer on thesolid support, and (4) performing high throughput electrosprayionization (ESI) or matrix-assisted laser desorption/ionization massspectrometry (HT MALDI MS) analysis on the captured peptides orproteins. Preferably, high throughput matrix-assisted laserdesorption/ionization time of flight mass spectrometry (HT MALDI TOF MS)analysis is performed.

In still another aspect of the invention, there is provided a method forperforming high throughput analysis of multiple samples, each of saidsamples comprising a mixture of peptides or proteins. The methodcomprises the steps of: (1) providing a solid support with a pluralityof compartments for sample application, wherein one or more of thecompartments has a specific affinity layer attached thereto for captureof the peptides or proteins present in the samples and wherein the oneor more compartments having a specific affinity layer attached theretofurther comprises a hydrophilic area devoid of and surrounded by thespecific affinity layer, (2) applying a sample or an aliquot thereof tothe one or more compartments having a specific affinity layer attachedthereto, (3) washing the peptides or proteins which have been capturedby the specific affinity layer on the solid support, and (4) performinghigh throughput electrospray ionization (ESI) or matrix-assisted laserdesorption/ionization mass spectrometry (HT MALDI MS) analysis on thecaptured peptides or proteins. Preferably, high throughputmatrix-assisted laser desorption/ionization time of flight massspectrometry (HT MALDI TOF MS) analysis is performed.

In yet a further embodiment, a method is provided for performing highthroughput analysis of multiple samples, wherein each of the samplescomprises a mixture of peptides or proteins. The method comprises thesteps of: (1) providing a solid support with a plurality of separatecompartments for sample application, wherein one or more of thecompartments has a specific affinity layer attached thereto for captureof the peptides or proteins present in the sample, wherein the specificaffinity layer is attached to a first portion of the one or morecompartments, wherein the first portion of the one or more compartmentscomprises a hydrophilic area devoid of and surrounded by the specificlayer attached thereto and wherein the one or more compartments furthercomprises a non-binding surface located in a second portion thereof, (2)applying a sample or an aliquot thereof to a separate compartment havinga specific affinity layer attached thereto; (3) washing the peptides orproteins which have been captured by the specific affinity layer, and(4) performing high throughput electrospray ionization (ESI) ormatrix-assisted laser desorption/ionization mass spectrometry (HT MALDIMS) analysis on the captured peptides or proteins. Preferably, highthroughput matrix-assisted laser desorption/ionization time of flightmass spectrometry (HT MALDI TOF MS) analysis is performed.

In any of the subject solid support compositions or methods using thesolid support compositions for performing high throughput analysis ofmultiple samples, it is preferable that more than one compartment has aspecific affinity layer attached thereto for protein and/or peptidecapture. Even more preferably, all or almost all of the compartmentshave a specific affinity layer attached thereto for protein and/orpeptide capture.

In accordance with the methods of the present invention, either anentire sample is applied or an aliquot of the sample is applied. Theterm “aliquot” as used herein means a portion of a sample but caninclude the entire sample in some instances especially if the samplesize is small. Typically, aliquots having anywhere from about a few atommoles to about 100 picomoles of proteinaceous material in a volume offrom about 1 μl to about 500 μl are applied to a single location(compartment) on the specific affinity layer of the solid support. Thesample is contacted to the specific affinity layer for a period of timesufficient to allow the peptides or proteins to bind to the specificaffinity layer. Typically, a sample is contacted with the specificaffinity layer for a period of time in the range of from about 30seconds to about 12 hours. Preferably, the sample is contacted with thespecific affinity layer for a period of time in the range of from about30 seconds to about 15 minutes.

Many different eluants or wash solutions may be used in the subjectmethods to remove unbound, non-proteinaceous material. For instance,pH-based eluants, which modify the selectivity of the specific affinitylayer based upon pH (charge), may be used and include known pH buffers,acidic solutions and basic solutions. Wash solutions that modify theselectivity of the specific affinity layer may also include ionicstrength-based eluants such as salt solutions of different types andconcentrations. Eluants, which modify the selectivity of the specificaffinity layer with respect to surface tension and protein or peptidestructure, include detergents and surfactants such as e.g., CHAPS,TWEEN, and NP-40. Eluants, which modify the selectivity of the specificaffinity layer based on hydrophobic interaction, include urea, organicsolvents such as propanol, acetonitrile, ethylene glycol and glycerol,and detergents.

Based on different parameters such as source of the peptide or proteinsample, and type of specific affinity layer attached to the solidsupport, one skilled in the art of chemical or biochemical analysis iscapable of determining the optimal wash conditions. For example, washesespecially suited for use on hydrophobic surface layers include urea,organic solvents and detergents. Eluants useful for washing peptides orproteins captured by ionic surface layers include dilute aqueousbuffers. Eluants useful for washing peptides or proteins captured bymetal chelating surfaces include metal chelating surfaces including, butnot limited to imidazole buffers, low pH or TRIS or other bufferincluding salts, e.g., sodium chloride and imidazole. Typically, theeluant is contacted to the specific affinity layer on the solid supportat a temperature in the range of from about 0° to 100° C. Preferably, awash temperature in the range of from about 4° to about 37° C. is used.

After washing the captured peptides or proteins, if MALDI MS is theanalytical tool of choice, the peptides or proteins are ultimatelycontained in a photo-excitable matrix. Examples of matrices which may beused in the methods of the invention include but are not limited todihydroxybenzoic acid (dhb) in acetonitrile, nicotinic acid, sinapinicacid, succinic acid, glycerol, alpha-cyano-4-hydroxycinnamic acid(HCCA), alpha-cyano-4-hydroxycinnamic-acid-methyl-ester (a-CNME),2,3,4-trihydroxyacetophenone (2,3,4-THAP), 2,4,6-trihydroxyacetophenone(2,4,6-THAP) and 3-hydroxypicolinic acid.

Conventional MALDI-MS equipment and methods may be applied. Commercialsystems are available such as e.g., HP 62025A MALDI-TOF (Hewlett-PackardCo., Palo Alto), PerSpective Biosystem Elite TOF, (PerSeptiveBiosystems, Framingham, Mass.) and IonSpec Ultima FT Mass Spectrometer(IonSpec, Irvine Calif.).

The invention is further illustrated by the following specific examples,which are not intended in any way to limit the scope of the invention.

EXAMPLE 1 Preparation of Octadecyl Vinyl Ether Coated Microwell Platesand Optimization of the Coating Process

Among the five compounds screened, octadecyl vinyl ether (ODVE) coatedplate (0.2%) showed highest protein capture tested by colloidal gold.Therefore ODVE was chosen for further testing by the FITC-BSA assay asthe most successful surface, especially ODVE coated on non-treatedpolystyrene. FIG. 2A reports the result of protein capture on untreatedpolystyrene surface.

ODVE was used as purchased from Aldrich Chemicals. The ODVE wasdissolved in isopropanol at a concentration range 0.1 to 1 weight %. Themicroplates were either Corning 384 well or Corning half-volume 96 wellplates tissue culture-treated polystyrene (TC). Coating was achieved byaddition of the coating solution to microplate wells followed by argonplasma treatment. The coating solution was added to wells 100 μl, andremoved after approximately 1 hr, dried for approximately 10 min. at 40°C. and then exposed to argon plasma in a Plasmod Etcher (MPS 300 RFPower Module with GCM-250 gas control module, March Instrument Inc.).The argon plasma was generated using argon at approximately 12 psi andin a vacuum of 0.8-1.0 torr. The treatment time range was 10 to 300 s ata RF power of 25 W.

To determine the optimal plasma exposure time, 1%, 0.5%, and 0.2% ODVEsolutions were coated onto non-treated polystyrene plates (96 well,half-volume). 100 μl of coating solution was added per well. Argonplasma exposure time was varied between 10 and 300 s. Coated plates werethen tested for protein capture and recovery using fluorescent-labeledBSA assay. 1 mg/ml fluorescein isothiocyanate-tagged BSA (FITC-BSA) in0.1% TFA/H₂O was added to coated microplate wells and left overnight.This solution was then removed and wells were washed twice with 0.1%TFA/H₂O. 70% acetonitrile was then added to selected wells (100 μl) toelute the bound protein. This solution was agitated by up and downpipetting and then removed. Wells were again washed twice with 0.1%TFA/H₂O. 0.1M Borate buffer was then added to the wells and fluorescencewas measured 30 min. later using a plate reader set to an excitationwavelength of 492 nm and an emission wavelength of 535 nm. Fluorescenceof capture wells (no acetonitrile addition) was compared to that ofcapture and recovery wells (acetonitrile addition) for each coatingconcentration and at each plasma exposure time (FIG. 2B). Allmeasurements were taken using a Perkin Elmer plate reader.

EXAMPLE 2

ODVE Coated Microwell Plates for MALDI TOF MS Analysis

To evaluate the utility of the C18-coated microwell plates, bovine serumalbumin (BSA) was digested with trypsin in a 100 mM NH₄HCO₃ solution (1mg/ml) overnight at 37° C. The BSA digest was diluted to desiredconcentration in 100 mM NH₄HCO₃. The tryptic digest was adsorbed byplacing 1-2111 of the digest into a well of the coated plate, andallowing the sample to be air-dried. Each well in the plate was washedtwice with 10 μl 0.1% TFA solution to desalt the peptides followed by 2μl of 50-75% acetonitrile water solution to extract the capturedpeptides. The MALDI TOF MS analysis consists of placing 1-2 μl of thepeptide solution in acetonitrile onto a stainless steel target plate andadding about 1 μl of matrix solution on target. The matrix used wasα-cyano-4-hydroxycinnamic acid (HCCA) obtained from Aldrich. HCCA wasdissolved in 70% acetonitrile at a concentration of approximately ˜10mg/ml. Mass spectra were acquired on the Bruker MALDI TOF BiflexIIIinstrument. Good MALDI TOF mass spectra were obtained after purificationof C18 coated plates as showed in the FIGS. 3A-C.

EXAMPLE 3 C18 On Gold Coated Microwell Plates

Gold was sputtered onto 384 polystyrene half-volume microplates. Theplates were coated with a solution of 1-octadecenethiol (ODAM) inalcohol to create a self-assembled monolayer (SAM) surface that cancapture proteins and peptides. Each well was loaded with ˜17 ul1-octadecanethiol alcohol solution (1 mM), after 30 minutes the platewere washed three times with alcohol, and air-dried. The tryptic BSAdigest was used to evaluate the C18 coated plate using the methodmentioned in example 2. The tryptic digest of BSA purified on the1-octadecenethiol coated plated gave a MALDI mass spectral datacomparable to ODVE coated plate (FIGS. 4A-F).

Although the present invention has been described generally and indetail by way of examples, persons skilled in the art will understandthat the invention is not limited to the embodiments specificallydisclosed, and that modifications and variations can be made withoutdeparting from the spirit and scope of the invention. Therefore, unlesschanges otherwise depart from the scope of the invention as defined bythe following claims, they should be construed as included herein.

REFERENCES

The following references and those cited throughout the specificationare incorporated herein by reference.

-   1. Naaby-Hansen, S: Waterfoe; d, M. D.; Cramer, R. Trends in    Pharmacological Sciences, 2001, 22, 376-384.-   2. Anderson, L.: Seilhamber, J. Electrophoresis 1997, 18, 533-537.-   3. Gygi, S. P. et al As a matter of law. Cell. Biol. 1999, 19,    1720-1730.-   4. Dove, A. Nature Biotechnology, 1999, 17 233-236.-   5. Edwards, A. M.: Arrowsmith, C. H.: Pallieres, B. Modern Drug    Discovery, 2000, 5(7), 35-44.-   6. Nature, 1999, 402, 715-719.-   7. Lee, K. H. Trends in Biotechnology, 2001, 19, 217-222.-   8. Fenn, J. B.: Mann, M.: Menig, C. K.; Wong, S. F.;    Whitehouse, C. M. Science, 1989, 246, 64.-   9. Cole, R. B.; Electrospray Ionization Mass Spectrometry:    Fundamentals, Instrumentation and Applications; Wiley: New York,    1997.-   10. Karas, M.; Hillenkam, F. Anal. Chem. 1995, 60, 2299.-   11. Loo, J. A. Bioconjugate Chem. 1995, 6, 644.-   12. Barber, M.; Mordoli, $. R.; Sedgwick, R., A. N. J. Chem. Soc.    Commun., 1981, 325.-   13. Aebersold, R.; Goodlett, D. R. Chem. Review. 2001, 101, 269-295.-   14. Chaurand, P.; Luetzenkirchen, F.; Spengler, B. J. Am. Soc. Mas.    Spectrom. 1999, 10, 91.-   15. Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem 1993, 65,    425-438.-   16. Langen, H.; Takacs, B.; Evers, S.; Bermdt, P.; Lahm, H. W.;    Wipf, B.; Gray, C.; Fountoulakis, M. Electrophoresis 2000, 21, 411.-   17. Naaby-Hansen, S; Waterford, M. D.; Cramer, R. Trends in    Pharmacological Sciences, 2001, 22, 376-384.-   18. Pluskal, M. G. Nature Biotechnology 2000, 18, 104-105.-   19. Watten, M. E.; Brockman, A. H.; Orlando, R. Anal. Chem. 1998,    70, 3757-3761.-   20. Beavis, R. C.; Chait, B. T.; Methods Enzymols. 1996, 270,    519-551.-   21. Wang, M. Z.; Fitzgerald, M. C. Anal. Chem. 2001, 73, 625-631.-   22. Zhang, N.; Doucette, A., Liability, L. Anal. Chem. 2001, 73,    2968-2975.-   23. Doucette, A.; Craft, D.; Liability, L. Anal. Chem. 2000, 72,    3355-3362.-   24. Warren, M. E.; Brockman A. H.; Orlando, R. Anal. Chem. 1998, 70,    3757-3761.-   25. Schurengerg, M. et al. Prestructured MALDI Sample Supports:    Sample Washing and Recrystallization, Bruker Daltonics; Bremen,    2001.-   26. Sharpe, L. H.; Schonhom, J. Adv. Chem. Ser. 1964, 4, 189-201.-   27. Hansen, R. H.; Schonhom, J. J. Polym. Sci. B, Plym. Lett. Ed.    1966, 4, 203-209.-   28. Sheu, M. S. et al. J. Adhesion Sci. Technol. 1993, 7, 1065-1076.-   29. Davies, J. et al. Colloids and Surfaces A: Physiochem. Eng.    Aspects 2000, 174, 287-295.

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled) 11.(canceled)
 12. (canceled)
 13. A method of performing high throughputanalysis of multiple samples, each of said samples comprising a mixtureof peptides or proteins, said method comprising the steps of: (a)providing a solid support comprising a plurality of separatecompartments, wherein one or more of the compartments has a specificaffinity layer attached thereto for capture of the peptides or proteinspresent in the samples; (b) applying each of said samples or an aliquotthereof, to a separate compartment having the specific affinity layerattached thereto; (c) washing the peptides or proteins which have beencaptured by the specific affinity layer, and (d) performing highthroughput electrospray ionization (ESI) or matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry analysis on the capturedpeptides or proteins.
 14. The method of claim 13 wherein the specificaffinity layer is attached to a first portion of the one or morecompartments and a non-binding surface is located in a second portion ofthe one or more compartments.
 15. The method of claim 13 wherein the oneor more compartments having a specific affinity layer attached theretofurther has a hydrophilic area devoid of and surrounded by the specificaffinity layer.
 16. The method of claim 13 wherein the specific affinitylayer is attached to a first portion of the one or more compartments,wherein the first portion of the one or more compartments comprises ahydrophilic area devoid of and surrounded by the specific affinity layerattached thereto and wherein the one or more compartments furthercomprises a non-binding surface located in a second portion thereof. 17.The method of any one of claims 13-16 wherein the specific affinitylayer is at least one of a hydrophobic surface layer, an ionic surfacelayer, or a metal chelating surface layer.
 18. The method of any one ofclaims 13-16 wherein the solid support is a dish, slide, strip or plate.19. The method of claim 18 wherein the solid support is a is a microwellplate or a microtiter dish.
 20. The method of any one of claims 13-16wherein the specific affinity layer is at least one of a hydrophobicsurface layer, an ionic surface layer, or a metal chelating surfacelayer.
 21. The method of claim 20 wherein the hydrophobic layercomprises a long chain aliphatic.
 22. The method of claim 21 wherein thelong chain aliphatic is at least one of octadecyl vinyl ether (ODVE),octadecyl amine (ODAM), tri(ethylene glycol)-methyl-vinyl-ether (TMVE),stearyl alcohol (SA), nonylphenoxypoly(ethyleneoxy)ethyl-methacrylate(C9 poly), or stearyloxypolyethyleneoxy-ethyl-methacrylate (SPMA). 23.The method of claim 20 wherein the metal chelating surface layer isnickel.
 24. The method of any one of claims 13-16 wherein the solidsubstrate comprises a polymer injection molded plate, a glass bottomplastic plate or a gold deposited plastic plate
 25. The method of claim15 or 16 wherein a photo-excitable matrix is added to the capturedpeptides or proteins in the one or more compartments.
 26. The method ofclaim 25 wherein the matrix is at least one of sinapinic acid,3-hydroxypicolinic acid, alpha-cyano-4-hydroxycinnamic acid,alpha-cyano-4-hydroxycinnamic acid methyl ester, 2,5-dihydroxybenzoicacid, 2,3,4-trihydroxyacetophenone, 2,4,6-trihydroxyacetophenone,alpha-cyano-4-hydroxycinnamic acid methyl ester (a-CNME),2,3,4-trihydroxyacetophenone (2,3,4-THAP), 2,4,6-trihydroxyacetophenone(2,4,6-THAP), nicotinic acid, succinic acid, or glycerol.